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Ca2+-permeable AMPA receptors (AMPARs) are expressed throughout the adult CNS but yet their role in development is poorly understood. In the developing retina, most investigations have focused on Ca2+ influx through NMDARs in promoting synapse maturation and not on AMPARs. However, NMDARs are absent from many retinal cells suggesting that other Ca2+-permeable glutamate receptors may be important to consider. Here we show that inhibitory horizontal and AII amacrine cells lack NMDARs but express Ca2+-permeable AMPARs. Before eye-opening, AMPARs were fully blocked by philanthotoxin (PhTX), a selective antagonist of Ca2+-permeable AMPARs. After eye-opening, however, a subpopulation of Ca2+-permeable AMPARs were unexpectedly PhTX resistant. Furthermore, Joro spider toxin (JSTX) and IEM-1460 also failed to antagonize, demonstrating that this novel pharmacology is shared by several AMPAR channel blockers. Interestingly, PhTX-insensitive AMPARs failed to express in retinae from dark-reared animals demonstrating that light entering the eye triggers their expression. Eye-opening coincides with the consolidation of inhibitory cell connections suggesting that the developmental switch to a Ca2+-permeable AMPAR with novel pharmacology may be critical to synapse maturation in the mammalian retina. [ABSTRACT FROM AUTHOR]

1. The action of philanthotoxin 343 (PhTX) on rat homomeric GluR6(Q) recombinant glutamate receptor channels was analysed using concentration-jump techniques and outside-out patches from HEK 293 cells. Both onset and recovery from block by external PhTX were dependent on the presence of agonist, indicating that channels must open for PhTX to bind and that channel closure can trap PhTX. 2. Block by external PhTX developed with double-exponential kinetics. The rate of onset of the fast component of block showed an exponential increase per 27 mV hyperpolarization over the range -40 to -100 mV. The rate of onset of the slow component of block showed a non-linear concentration dependence indicating a rate-limiting step in the blocking mechanism. 3. The extent of block by 1 microM external PhTX was maximal at -40 mV and did not increase with further hyperpolarization; the rate of recovery from block by external PhTX increased 6-fold on hyperpolarization from -40 to -100 mV suggesting that PhTX permeates at negative membrane potentials. 4. Apparent Kd values for block by external PhTX estimated from dose-inhibition experiments decreased 300-fold on hyperpolarization from +40 mV (Kd, 19.6 microM) to -40 mV (Kd, 69 nM); there was little further increase in affinity with hyperpolarization to -80 mV (Kd, 56 nM), consistent with permeation of PhTX at negative membrane potentials. 5. Block by internal PhTX showed complex kinetics and voltage dependence. Analysis with voltage ramps from -120 to +120 mV indicated a Kd at 0 mV of 20 microM, decreasing e-fold per 16 mV depolarization. However, at +90 mV the extent of block by 1 and 10 microM internal PhTX (73 % and 95 %, respectively) reached a maximum and did not increase with further depolarization. 6. Voltage-jump analysis of block by 100 microM internal PhTX revealed partial trapping. With 100 ms jumps from -100 to -40 mV, onset and recovery from block were complete within 5 ms. With jumps of longer duration the extent of block increased, with a time constant of 8.1 s, reaching 84 % at 30 s. On repolarization to -100 mV, recovery from block showed fast and slow components. 7. The amplitude of the slow component of block by internal PhTX showed a biphasic voltage dependence, first increasing then decreasing with progressive depolarization. Maximum block was obtained at 0 mV. 8. Our results suggest that PhTX acts as an open channel blocker; however, provided that the toxin remains bound to the channel, an allosteric mechanism destabilizes the open state, inducing channel closing and trapping PhTX. Strong depolarization for internal PhTX, or strong hyperpolarization for external PhTX, forces the toxin to permeate before it triggers entry into closed blocked states.

The human TE671 cell line was originally used as a model of medulloblastoma but has since been reassigned as rhabdomyosarcoma. Despite the characterised endogenous expression of voltage‐sensitive sodium currents in these cells, the specific voltage‐gated sodium channel (VGSC) subtype underlying these currents remains unknown. To profile the VGSC subtype in undifferentiated TE671 cells, endpoint and quantitative reverse transcription–PCR (qRT‐PCR), western blot and whole‐cell patch clamp electrophysiology were performed. qRT‐PCR profiling revealed that expression of the SCN9A gene was ∼215‐fold greater than the SCN4A gene and over 400‐fold greater than any of the other VGSC genes, while western blot confirmed that the dominant SCN9A RNA was translated to a protein with a molecular mass of ∼250 kDa. Elicited sodium currents had a mean amplitude of 2.6 ± 0.7 nA with activation and fast inactivation V50 values of −31.9 ± 1.1 and −69.6 ± 1.0 mV, respectively. The currents were completely and reversibly blocked by tetrodotoxin at concentrations greater than 100 nm (IC50 = 22.3 nm). They were also very susceptible to the NaV1.7 specific blockers Huwentoxin‐IV and Protoxin‐II with IC50 values of 14.6 nm and 0.8 nm, respectively, characteristic of those previously determined for NaV1.7. Combined, the results revealed the non‐canonical and highly dominant expression of NaV1.7 in the human TE671 rhabdomyosarcoma cell line. We show that the TE671 cell line is an easy to maintain and cost‐effective model for the study of NaV1.7, a major target for the development of analgesic drugs and more generally for the study of pain. Key points: Undifferentiated TE671 cells produce a voltage‐sensitive sodium current when depolarised.The voltage‐gated sodium channel isoform expressed in undifferentiated TE671 cells was previously unknown. Through qRT‐PCR, western blot and toxin pharmacology, it is shown that undifferentiated TE671 cells dominantly (>99.5%) express the NaV1.7 isoform that is strongly associated with pain. The TE671 cell line is, therefore, a very easy to maintain and cost‐effective model to study NaV1.7‐targeting drugs. [ABSTRACT FROM AUTHOR]

Intentional ingestion of agricultural organophosphorus insecticides is a significant public health issue in rural Asia, causing thousands of deaths annually. Some survivors develop a severe, acute or delayed myasthenic syndrome. In animal models, similar myasthenia has been associated with increasing plasma concentration of one insecticide solvent metabolite, cyclohexanol. We investigated possible mechanisms using voltage and current recordings from mouse neuromuscular junctions (NMJs) and transfected human cell lines. Cyclohexanol (10–25 mM) reduced endplate potential (EPP) amplitudes by 10–40% and enhanced depression during repetitive (2–20 Hz) stimulation by up to 60%. EPP decay was prolonged more than twofold. Miniature EPPs were attenuated by more than 50%. Cyclohexanol inhibited whole‐cell currents recorded from CN21 cells expressing human postjunctional acetylcholine receptors (hnAChR) with an IC50 of 3.74 mM. Cyclohexanol (10–20 mM) also caused prolonged episodes of reduced‐current, multi‐channel bursting in outside‐out patch recordings from hnAChRs expressed in transfected HEK293T cells, reducing charge transfer by more than 50%. Molecular modelling indicated cyclohexanol binding (−6 kcal/mol) to a previously identified alcohol binding site on nicotinic AChR α‐subunits. Cyclohexanol also increased quantal content of evoked transmitter release by ∼50%. In perineurial recordings, cyclohexanol selectively inhibited presynaptic K+ currents. Modelling indicated cyclohexanol binding (−3.8 kcal/mol) to voltage‐sensitive K+ channels at the same site as tetraethylammonium (TEA). TEA (10 mM) blocked K+ channels more effectively than cyclohexanol but EPPs were more prolonged in 20 mM cyclohexanol. The results explain the pattern of neuromuscular dysfunction following ingestion of organophosphorus insecticides containing cyclohexanol precursors and suggest that cyclohexanol may facilitate investigation of mechanisms regulating synaptic strength at NMJs. Key points: Intentional ingestion of agricultural organophosphorus insecticides is a significant public health issue in rural Asia, causing thousands of deaths annually. Survivors may develop a severe myasthenic syndrome or paralysis, associated with increased plasma levels of cyclohexanol, an insecticide solvent metabolite.Analysis of synaptic transmission at neuromuscular junctions in isolated mouse skeletal muscle, using isometric tension recording and microelectrode recording of endplate voltages and currents, showed that cyclohexanol reduced postsynaptic sensitivity to acetylcholine neurotransmitter (reduced quantal size) while simultaneously enhancing evoked transmitter release (increased quantal content).Patch recording from transfected cell lines, together with molecular modelling, indicated that cyclohexanol causes selective, allosteric antagonism of postsynaptic nicotinic acetylcholine receptors and block of presynaptic K+‐channel function.The data provide insight into the cellular and molecular mechanisms of neuromuscular weakness following intentional ingestion of agricultural organophosphorus insecticides. Our findings also extend understanding of the effects of alcohols on synaptic transmission and homeostatic synaptic function. [ABSTRACT FROM AUTHOR]

AMPA receptors are tetrameric glutamate‐gated ion channels that mediate a majority of fast excitatory neurotransmission in the brain. They exist as calcium‐impermeable (CI‐) and calcium‐permeable (CP‐) subtypes, the latter of which lacks the GluA2 subunit. CP‐AMPARs display an array of distinctive biophysical and pharmacological properties that allow them to be functionally identified. This has revealed that they play crucial roles in diverse forms of central synaptic plasticity. Here we summarise the functional hallmarks of CP‐AMPARs and describe how these are modified by the presence of auxiliary subunits that have emerged as pivotal regulators of AMPARs. A lasting change in the prevalence of GluA2‐containing AMPARs, and hence in the fraction of CP‐AMPARs, is a feature in many maladaptive forms of synaptic plasticity and neurological disorders. These include modifications of glutamatergic transmission induced by inflammatory pain, fear conditioning, cocaine exposure, and anoxia‐induced damage in neurons and glia. Furthermore, defective RNA editing of GluA2 can cause altered expression of CP‐AMPARs and is implicated in motor neuron damage (amyotrophic lateral sclerosis) and the proliferation of cells in malignant gliomas. A number of the players involved in CP‐AMPAR regulation have been identified, providing useful insight into interventions that may prevent the aberrant CP‐AMPAR expression. Furthermore, recent molecular and pharmacological developments, particularly the discovery of TARP subtype‐selective drugs, offer the exciting potential to modify some of the harmful effects of increased CP‐AMPAR prevalence in a brain region‐specific manner. [ABSTRACT FROM AUTHOR]

AMPA‐type glutamate receptors (AMPARs) are key molecules of neuronal communication in our brain. The discovery of AMPAR auxiliary subunits, such as proteins of the TARP, CKAMP and CNIH families, fundamentally changed our understanding of how AMPAR function is regulated. Auxiliary subunits control almost all aspects of AMPAR function in the brain. They influence AMPAR assembly, composition, structure, trafficking, subcellular localization and gating. This influence has important implications for synapse function. In the present review, we first discuss how auxiliary subunits affect the strength of synapses by modulating number and localization of AMPARs in synapses as well as their glutamate affinity, conductance and peak open probability. Next we explain how the presence of auxiliary subunits alters temporal precision and integrative properties of synapses by influencing gating kinetics of the receptors. Auxiliary subunits of the TARP and CKAMP family modulate synaptic short‐term plasticity by increasing anchoring of AMPARs in synapses and by altering their desensitization kinetics. We then describe how auxiliary subunits of the TARP, CKAMP and CNIH families are involved in Hebbian and homeostatic plasticity, which can be explained by their influence on surface trafficking and synaptic targeting. In conclusion, the series of studies covered in this review show that auxiliary subunits play a pivotal role in controlling information processing in the brain by modulating synaptic computation. [ABSTRACT FROM AUTHOR]

Key points Large premotor neurons of the cerebellar nuclei (CbN cells) integrate synaptic inhibition from Purkinje neurons and synaptic excitation from mossy fibres to generate cerebellar output., We find that mossy fibre inputs to CbN cells generate unitary AMPA receptor EPSCs of ∼1 nS that decay in ∼1 ms and mildly voltage-dependent NMDA receptor EPSCs of ∼0.6 nS that decay in ∼7 ms., A few hundred mossy fibres active at a few tens of spikes s−1 must converge on CbN cells to generate physiological CbN spike rates (∼60 spikes s−1) during convergent inhibition from spontaneously active Purkinje cells., Dynamic clamp studies in cerebellar slices from weanling mice demonstrate that synaptic excitation from mossy fibres becomes more effective at increasing the rate of CbN cell spiking when the coherence (synchrony) of convergent inhibition is increased., Abstract Large projection neurons of the cerebellar nuclei (CbN cells), whose activity generates movement, are inhibited by Purkinje cells and excited by mossy fibres. The high convergence, firing rates and strength of Purkinje inputs predict powerful suppression of CbN cell spiking, raising the question of what activity patterns favour excitation over inhibition. Recording from CbN cells at near-physiological temperatures in cerebellar slices from weanling mice, we measured the amplitude, kinetics, voltage dependence and short-term plasticity of mossy fibre-mediated EPSCs. Unitary EPSCs were small and brief (AMPA receptor, ∼1 nS, ∼1 ms; NMDA receptor, ∼0.6 nS, ∼7 ms) and depressed moderately. Using these experimentally measured parameters, we applied combinations of excitation and inhibition to CbN cells with dynamic clamp. Because Purkinje cells can fire coincident simple spikes during cerebellar behaviours, we varied the proportion (0-20 of 40) and precision (0-4 ms jitter) of synchrony of inhibitory inputs, along with the rates (0-100 spikes s−1) and number (0-800) of excitatory inputs. Even with inhibition constant, when inhibitory synchrony was higher, excitation increased CbN cell firing rates more effectively. Partial inhibitory synchrony also dictated CbN cell spike timing, even with physiological rates of excitation. These effects were present with ≥10 inhibitory inputs active within 2-4 ms of each other. Conversely, spiking was most effectively suppressed when inhibition was maximally asynchronous. Thus, the rate and relative timing of Purkinje-mediated inhibition set the rate and timing of cerebellar output. The results suggest that increased coherence of Purkinje cell activity can facilitate mossy fibre-driven spiking by CbN cells, in turn driving movements. [ABSTRACT FROM AUTHOR]

Glutamate receptor-mediated recruitment of GABAergic inhibitory interneurons is a critical determinant of network processing. Early studies observed that many, but not all, interneuron glutamatergic synapses contain AMPA receptors that are GluA2-subunit lacking and Ca2+ permeable, making them distinct from AMPA receptors at most principal cell synapses. Subsequent studies demonstrated considerable alignment of synaptic AMPA and NMDA receptor subunit composition within specific subtypes of interneurons, suggesting that both receptor expression profiles are developmentally and functionally linked. Indeed glutamate receptor expression profiles are largely predicted by the embryonic origins of cortical interneurons within the medial and caudal ganglionic eminences of the developing telencephalon. Distinct complements of AMPA and NMDA receptors within different interneuron subpopulations contribute to the differential recruitment of functionally divergent interneuron subtypes by common afferent inputs for appropriate feed-forward and feedback inhibitory drive and network entrainment. In contrast, the lesser-studied kainate receptors, which are often present at both pre- and postsynaptic sites, appear to follow an independent developmental expression profile. Loss of specific ionotropic glutamate receptor (iGluR) subunits during interneuron development has dramatic consequences for both cellular and network function, often precipitating circuit inhibition-excitation imbalances and in some cases lethality. Here we briefly review recent findings highlighting the roles of iGluRs in interneuron development. [ABSTRACT FROM AUTHOR]

Key points In the hippocampus, calcium-permeable AMPA receptors have been found in a restricted subset of neuronal types that inhibit other neurons, although their localization in the neocortex is less well understood., In the present study, we looked for calcium-permeable AMPA receptors in two distinct populations of neocortical inhibitory neurons: basket cells and Martinotti cells. We found them in the former but not in the latter. Furthermore, in basket cells, these receptors were associated with particularly fast responses., Computer modelling predicted (and experiments verified) that fast calcium-permeable AMPA receptors enable basket cells to respond rapidly, such that they promptly inhibit neighbouring cells and shut down activity., The results obtained in the present study help our understanding of pathologies such as stroke and epilepsy that have been associated with disordered regulation of calcium-permeable AMPA receptors., Abstract AMPA-type glutamate receptors (AMPARs) lacking an edited GluA2 subunit are calcium-permeable (CP) and contribute to synaptic plasticity in several hippocampal interneuron types, although their precise role in the neocortex is not well described. We explored the presence of CP-AMPARs at pyramidal cell (PC) inputs to Martinotti cells (MCs) and basket cells (BCs) in layer 5 of the developing mouse visual cortex (postnatal days 12-21). GluA2 immunolabelling was stronger in MCs than in BCs. A differential presence of CP-AMPARs at PC-BC and PC-MC synapses was confirmed electrophysiologically, based on measures of spermine-dependent rectification and CP-AMPAR blockade by 1-naphtyl acetyl spermine using recordings from synaptically connected cell pairs, NPEC-AMPA uncaging and miniature current recordings. In addition, CP-AMPAR expression in BCs was correlated with rapidly decaying synaptic currents. Computer modelling predicted that this reduces spike latencies and sharpens suprathreshold responses in BCs, which we verified experimentally using the dynamic clamp technique. Thus, the synapse-specific expression of CP-AMPARs may critically influence both plasticity and information processing in neocortical microcircuits. [ABSTRACT FROM AUTHOR]

Cortical neuronal network operations depend critically on the recruitment of GABAergic interneurons and the properties of their inhibitory output signals. Recent evidence indicates a marked difference in the signalling properties of two major types of perisomatic inhibitory interneurons, the parvalbumin- and the cholecystokinin-containing basket cells. Parvalbumin-expressing basket cells are rapidly recruited by excitatory synaptic inputs, generate high-frequency trains of action potentials, discharge single action potentials phase-locked to fast network oscillations and provide fast, stable and timed inhibitory output onto their target cells. In contrast, cholecystokinin-containing basket cells are recruited in a less reliable manner, discharge at moderate frequencies with single action potentials weakly coupled to the phases of fast network oscillations and generate an asynchronous, fluctuating and less timed inhibitory output. These signalling modes are based on cell type-dependent differences in the functional and plastic properties of excitatory input synapses, integrative qualities and in the kinetics and dynamics of inhibitory output synapses. Thus, the two perisomatic inhibitory interneuron types operate with different speed and precision and may therefore contribute differently to the operations of neuronal networks. [ABSTRACT FROM AUTHOR]

The subunit composition of synaptic AMPA receptors can undergo dynamic changes during physiological functioning and under pathological conditions. This switch in AMPA receptor phenotype involves changes in the level of GluA2 subunits that are mediated via regulated AMPA receptor trafficking, modification of local protein synthesis and altered gene transcription of GluA2 subunits. Incorporation of the GluA2 subunits into an AMPA receptor alters a number of key biophysical properties, including Ca2+ permeability and the waveform of the synaptic current. These changes alter the ability of synaptic currents to evoke an action potential and therefore have a profound effect on the computational capability of individual neurons and thus the output of neuronal circuits. [ABSTRACT FROM AUTHOR]

AMPA-type ionotropic glutamate receptors (iGluRs) represent the major excitatory neurotransmitter receptor in the developing and adult vertebrate CNS. They are crucial for the normal hardwiring of glutamatergic circuits but also fine tune synaptic strength by cycling into and out of synapses during periods of sustained patterned activity or altered homeostasis. AMPARs are grouped into two functionally distinct tetrameric assemblies based on the inclusion or exclusion of the GluA2 receptor subunit. GluA2-containing receptors are thought to be the most abundant AMPAR in the CNS, typified by their small unitary events, Ca2+ impermeability and insensitivity to polyamine block. In contrast, GluA2-lacking AMPARs exhibit large unitary conductance, marked divalent permeability and nano- to micromolar polyamine affinity. Here, I review evidence for the existence of a third class of AMPAR which, though similarly Ca2+ permeable, is characterized by its near-insensitivity to internal and external channel block by polyamines. This novel class of AMPAR is most notably found at multivesicular release synapses found in the avian auditory brainstem and mammalian retina. Curiously, these synapses lack NMDA-type iGluRs, which are conventionally associated with controlling AMPAR insertion. The lack of NMDARs suggests that a different set of rules may govern AMPAR cycling at these synapses. AMPARs with similar functional profiles are also found on some glial cells suggesting they may have a more widespread distribution in the mammalian CNS. I conclude by noting that modest changes to the ion-permeation pathway might be sufficient to retain divalent permeability whilst eliminating polyamine sensitivity. Consequently, this emerging AMPAR subclass need not be assembled from novel subunits, yet to be cloned, but could simply occur by varying the stoichiometry of existing proteins. [ABSTRACT FROM AUTHOR]

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Hebbian long-term potentiation (LTP) develops at specific synapses onto hippocampal CA1 oriens/alveus interneurons (OA-INs), suggesting selective regulation of distinct input pathways. Afferent-specific properties at interneuron synapses have been characterized extensively in CA3 stratum lucidum cells, but given interneuron diversity these rules of transmission and plasticity may not hold in other interneuron types. Here, we used paired recordings and demonstrate that CA2/3 pyramidal cell (PC) feedforward and CA1 PC feedback synapses onto OA-INs show distinct AMPA receptor rectification and Ca2+ permeability, short-term plasticity and mGluR2/3-mediated inhibition. Only feedback synapses undergo Hebbian LTP. OA-IN firing during repeated synaptic stimulation displays onset-transient or late-persistent responses consistent with activation of feedforward and feedback inputs, respectively. Input-output functions are preserved after theta-burst stimulation, but late-persistent responses selectively show mGluR1-dependent long-term increases. Thus, cell type- and afferent-specific rules of transmission and plasticity underlie distinct OA-IN input-output functions, providing selective long-term regulation in feedback inhibitory networks. [ABSTRACT FROM AUTHOR]

Many synapses undergo immediate and persistent activity-dependent changes in strength via processes that fall under the umbrella of synaptic plasticity. It is known that this type of synaptic plasticity exhibits an underlying state dependence; that is, as synapses change in strength they move into distinct ‘states’ that are defined by the mechanism and ability to undergo future plasticity. In this study, we have investigated the molecular mechanisms that underlie state-dependent synaptic plasticity. Using intracellular application of peptides that mimic the C-terminal tail sequences of GluR1 and GluR2 AMPA receptor subtypes, combined with paired recordings of minimal synaptic connections, we have shown that AMPA receptor subtypes present in the membrane at a given time confer some properties of plasticity states. These data show that during synaptic plasticity, AMPA receptor subtypes are differentially stabilized by postsynaptic density proteins in or out of the postsynaptic membrane, and this differential synaptic expression of different AMPA receptor subtypes defines distinct synaptic states. [ABSTRACT FROM AUTHOR]

A rise in intracellular calcium levels ([Ca2+]i) is a key trigger for the lethal effects of the excitatory neurotransmitter glutamate in various central neurons, but a consensus has not been reached on the pathways that mediate glutamate-dependent increases of [Ca2+]i in retinal ganglion cells (RGCs). Using Ca2+ imaging techniques we demonstrated that, in the absence of external Mg2+, the Ca2+ signal evoked by glutamate was predominantly mediated by NMDA-type glutamate receptors (NMDA-Rs) in immunopanned RGCs isolated from neonatal or adult rats. Voltage-gated Ca2+ channels and AMPA/kainate-Rs contributed a smaller portion of the Ca2+ response at saturating concentrations of glutamate. Consistent with NMDA-R involvement, extracellular Mg2+ inhibited RGC glutamate responses, while glycine had a potentiating effect. With Mg2+ present externally, the effect of AMPA/kainate-R antagonists was enhanced and both NMDA- and AMPA/kainate-R antagonists greatly reduced the glutamate-induced increases of RGC [Ca2+]i. This finding indicates that the primary contribution of AMPA/kainate-Rs to RGC glutamatergic Ca2+ dynamics is through the depolarization-dependent relief of the Mg2+ block of NMDA-R channels. The effect of glutamate receptor antagonists on glutamatergic Ca2+ signals from RGCs in adult rat retinal wholemounts yielded results similar to those obtained using immunopanned RGCs. Additional experiments on isolated RGCs revealed that during a 1 h glutamate (10–1000 μm) exposure, 18–28% of RGCs exhibited delayed Ca2+ deregulation (DCD) and the RGCs that underwent DCD were positive for the death marker annexin V. RGCs with larger glutamate-evoked Ca2+ signals were more likely to undergo DCD, and NMDA-R blockade significantly reduced the occurrence of DCD. Identifying the mechanisms underlying RGC excitotoxicity aids in our understanding of the pathophysiology of retinal ischaemia, and this work establishes a major role for NMDA-R-mediated increases in [Ca2+]i in glutamate-related RGC death. [ABSTRACT FROM AUTHOR]

AMPA receptors (AMPARs) mediate the bulk of fast synaptic excitation in the CNS. We have recently shown that AMPAR-dependent synaptic transmission in immature neocortical pyramidal neurons is mediated by GluR2-deficient receptors that can be modulated by intra- or extracellular polyamines (PAs). Phosphorylation of AMPARs, e.g. by PKC, can lead to enhanced excitation, and PAs are known to modulate PKC activity. Therefore, PAs and PKC might interact to influence AMPAR function. To test this hypothesis, we made whole cell recordings from immature (P12–14) layer V pyramidal neurons and assayed two measures of PA influence on synaptic AMPAR function – inward rectification and use-dependent unblock (UDU), with the latter assayed by differences in rectification between a pair of EPSCs evoked at short (50 ms) latencies. We have previously shown that EPSCs in immature pyramidal neurons displayed inward rectification, which was enhanced by intracellular spermine, as was UDU. Staurosporin (ST), a PKC inhibitor, reversed the effect of PA on rectification and UDU, suggesting that PKC modulates postsynaptic activation of AMPARs. Similarly, polyamine-dependent rectification of spontaneous EPSCs was reversed by treatment with ST or GFX109203X, a specific PKC inhibitor. Chelating intracellular Ca2+ with BAPTA reproduced the effects of ST. In addition, PA immunoreactivity in layer V pyramidal neurons was reduced by PKC inhibition indicating that PKC activity influences PA metabolism. Taken together, these data support the involvement of postsynaptic PKC activation in both the inward rectification and UDU of EPSCs in immature rat cortex, and suggest an important mechanism by which excitatory synaptic transmission can be dynamically modulated by changes in either [Ca2+]i or [PA]i. [ABSTRACT FROM AUTHOR]

The agonist-binding domain of ionotropic glutamate receptors (iGluRs) has recently been crystallized as two polypeptide chains with a linker region. Although work on the structure of this isolated ligand-binding core has been invaluable, there is debate over how it relates to conformations adopted by intact receptors. iGluR crystals are proposed to represent the activated state as their degree of domain closure correlates well with agonist efficacy. However, iGluR crystals exhibit high agonist affinity that more closely matches that of desensitized receptors. Consequently, conformations adopted by iGluR crystals may represent this state. To test this, we have employed the plant lectin, concanavalin-A (Con-A) to report conformational changes elicited by kainate (KA) iGluR agonists during desensitization. When GluR6 KA receptors (KARs) were pre-incubated with Con-A, equilibrium responses evoked by the full agonist,l-glutamate (l-Glu), increased almost 30-fold. However, in the continued presence ofl-Glu, Con-A exerted no effect suggesting that it has restricted access to its binding sites when the agonist is bound. However, Con-A does not discriminate well between agonist-bound or -unbound states with the weak partial agonist, domoate. Accessibility experiments with KA were intermediate in nature consistent with its equilibrium efficacy at GluR6 KARs. Our results suggest that full and partial agonists elicit distinct conformational changes in KARs during desensitization. This finding can be reconciled with crystallographic data if the agonist-binding domain adopts the same conformation in the activated and desensitized states. However, other interpretations are possible suggesting future work is required if this issue is to be resolved. [ABSTRACT FROM AUTHOR]

An introduction to the journal is presented in which the editor discusses various reports published within the issue, including one on the effect of fear-inducing stimulus GluA2 AMPA-type ionotrophic glutamate receptor gene, one on the effects of reactive oxygen species of neuronal nicotinic acetylcholine receptors to dysautonomias in patients with diabetics, and another on the role of transient receptor potential channels in osmo- and thermoregulation.